Coronavirus modulation of cellular microRNAs in the liver: role in hepatitis

冠状病毒对肝脏细胞 microRNA 的调节：在肝炎中的作用

• 批准号: 8113557
• 负责人: SONIA NAVAS-MARTIN
• 金额: $ 23.18万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-29 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8113557
• 关键词: Acute; Acute Hepatitis; Address; Animals; Binding Sites; Biological; Biological Assay; Cell physiology; Cells; Complex; Computer Simulation; Coronavirus; Coronavirus Infections; DNA Viruses; Data; Disease; Disease Outcome; Elements; Endothelial Cells; Experimental Models; Functional RNA; Gene Expression; Genes; Genome; Health; Hepatic; Hepatitis; Hepatitis B; Hepatitis C; Hepatocyte; Human; Immune system; In Vitro; Infection; Inflammation; Inflammatory; Interferon Type II; Interferons; Intervention; Investigation; Knowledge; Kupffer Cells; Liver; Liver diseases; Luciferases; Mediating; Messenger RNA; MicroRNAs; Microarray Analysis; Modeling; Molecular; Molecular Profiling; Murine hepatitis virus; Mus; Nuclear; Outcome; Pathogenesis; Pattern; Platelet Factor 4; Play; Poly(A)+ RNA; RNA Viruses; Regulation; Regulatory T-Lymphocyte; Relative (related person); Reporter; Reverse Transcriptase Polymerase Chain Reaction; Role; Small RNA; System; Testing; Therapeutic; Time; Translational Repression; Translations; Tropism; Untranslated Regions; Validation; Viral; Viral Genes; Viral hepatitis; Virus; Virus Diseases; base; cellular targeting; cytokine; human coronavirus; in vivo; innovation; knock-down; liver function; mRNA Transcript Degradation; macrophage; man; novel; pathogen; positional cloning; promoter; prototype; respiratory; response; transcription factor; virus host interaction

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are small RNAs that regulate gene expression by translational repression and/or mRNA degradation. miRNAs may also up-regulate translation under certain circumstances. There is increasing evidence indicating that miRNAs play a major role in fundamental cellular processes, the regulation of the immune system, as well as in the onset and progression of many diseases. The role of miRNAs in virus-host interaction is just starting to be addressed. miRNAs have been found in some viruses and miRNAs from the host cell may play a role in regulating viral genes and even determine viral tropism. We have investigated whether coronaviruses (CoVs), a group of emerging animal and human RNA viruses, may have the ability to modulate the expression of some cellular miRNAs, which may determine disease outcome. Using a reverse genetic system to manipulate the CoV genome, we have previously characterized the molecular determinants of CoV-induced hepatitis, showed that the ability of murine coronaviruses to induce hepatitis is virus strain specific, and identified cellular targets in the liver (hepatocytes, endothelial cells, and the resident macrophage Kupffer cells). In Preliminary Studies, we have used miRNA microarray analysis and validation by real-time RTPCR to provide evidence of the differential expression of selected cellular miRNAs in macrophages upon in vitro infection with various strains of murine CoVs that induce different hepatitis outcome in the mouse (from minimal inflammation to fulminant hepatitis). Changes in the macrophage miRNAs profiles after murine CoVs infection were overlapping but distinct, and were observed early after infection in the absence of type I IFNs, IFN-?, and pro-inflammatory cytokine secretion. Therefore, we hypothesized that early alteration of the miRNAs expression profile in the CoV-infected mouse liver is a consequence of virus infection, and that strain-specific changes in miRNAs expression contribute to the differences in hepatitis outcome. Thus, we propose the following Specific Aims: (1) Using microarray analysis and real time RT-PCR, we will determine alterations in miRNA profiles in the mouse liver and in primary liver cells (hepatocytes and Kupffer cells) freshly isolated from infected mice, which are different between murine CoVs that differ in hepatitis outcome (MHV-A59, acute moderate hepatitis; and MHV-3, fulminant hepatitis); (2) Based upon results in Specific Aim 1, we will subsequently identify potential cellular miRNAs targets in 3'UTRs by in silico and functional approaches, focusing on those miRNAs whose expression is differentially modulated (up or down) between the viruses listed above and might be involved in fulminant hepatitis; and, (3) We will define the biological effects of knocking-down selected miRNAs (identified in the previous aims) in Kupffer and regulatory T cells and determine the role that those selected miRNAs may have in CoV-induced hepatitis outcome by specific in vivo miRNA-silencing using antagomirs.

描述（由申请人提供）：microRNA（miRNA）是小的RNA，可通过翻译抑制和/或mRNA降解来调节基因表达。在某些情况下，miRNA也可能上调翻译。越来越多的证据表明，miRNA在基本细胞过程，免疫系统的调节以及许多疾病的发作和进展中起着重要作用。 miRNA在病毒宿主相互作用中的作用才刚刚开始解决。在某些病毒中发现了miRNA，宿主细胞中的miRNA可能在调节病毒基因，甚至确定病毒性向流中起作用。我们已经调查了冠状病毒（COVS）是一组新兴的动物和人RNA病毒，可能具有调节某些细胞miRNA的表达的能力，这可能决定疾病的结果。 Using a reverse genetic system to manipulate the CoV genome, we have previously characterized the molecular determinants of CoV-induced hepatitis, showed that the ability of murine coronaviruses to induce hepatitis is virus strain specific, and identified cellular targets in the liver (hepatocytes, endothelial cells, and the resident macrophage Kupffer cells).在初步研究中，我们已经使用miRNA微阵列分析和实时RTPCR验证来提供巨噬细胞中选定细胞miRNA的差异表达的证据，并在体外感染中具有各种鼠COV菌株的体外感染，从而诱导小鼠中不同的肝炎结果（从最小的炎症到最小炎症到氟肝炎）。鼠Covs感染后巨噬细胞miRNA轮廓的变化是重叠但截然不同的，并且在没有I型IFN，IFN-？和促炎性细胞因子分泌的情况下感染了早期。因此，我们假设在COV感染的小鼠肝脏中miRNA表达谱的早期改变是病毒感染的结果，而miRNAS表达的菌株特异性变化导致肝炎预后的差异。 Thus, we propose the following Specific Aims: (1) Using microarray analysis and real time RT-PCR, we will determine alterations in miRNA profiles in the mouse liver and in primary liver cells (hepatocytes and Kupffer cells) freshly isolated from infected mice, which are different between murine CoVs that differ in hepatitis outcome (MHV-A59, acute moderate hepatitis; and MHV-3,暴发性肝炎）； （2）基于特定目标1中的结果，我们将通过在3'UTR中通过计算机和功能方法确定3'UTR中的潜在细胞miRNA靶标，重点是那些在上面列出的病毒之间进行差异调节（向上或向下）的miRNA，并可能参与funminant肝炎； （3）我们将在库普弗（Kupffer）和调节性T细胞中定义敲除选定的miRNA（在上一个目标中鉴定）的生物学作用，并确定所选miRNA在COV诱导的肝炎结局中通过使用Antagomirs进行特定的体内miRNA-lemnence的作用。